Dopamine (DA) antibody

Details for Product No. ABIN6559946
Antigen
Reactivity
Chemical
27
5
2
Host
Rabbit
27
19
Clonality
Polyclonal
Conjugate
Un-conjugated
4
4
1
1
1
1
Application
Immunocytochemistry (ICC)
22
15
11
11
9
7
3
3
2
1
1
1
Options
Immunogen Synthetic dopamine conjugated to protein carrier (Pc)
Isotype IgG
Specificity Conjugated dopamine . Using a conjugate dopamine-(Pc), antibody specificity was performed with an ELISA test by competition experiments with the following compounds: Compounds Cross-reactivity ratio (a) Dopamine-G-(Pc) 1 Noradrenaline-G-(Pc) 1/200 L-DOPA-G-(Pc) 1/400 Octopamine-G-(Pc) 1/400 Tyramine-G-(Pc) 1/>5,000 Adrenaline-G-(Pc) 1/>5,000 Free Dopamine 1/>5,000 (a): Dopamine-G-(Pc) concentration/unconjugated or conjugated catecholamine concentration at half displacement G = Glutaraldehyde
Purification Antiserum previously preabsorbed on protein carriers, and purified
Antigen
Alternative Name Dopamine
Target Type Chemical
Application Notes Example of cytochemical application Detection of conjugated Dopamine in rat brain 1- Perfusion: The rat is anesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the following solutions: Solution A: cacodylate 0.1M, sodium metabisulfite 10g/l, pH = 6.2 Solution B: cacodylate 0.1M, sodium metabisulfite 10g/l and glutaraldehyde 3-5%, pH = 7.5 2- Post fixation: 15 to 30 min in solution B, then 4 soft washes in Tris 0.05M with sodium metabisulfite 8.5g/l, pH 7.5 (solution C). 3- Tissue sectionning: Cryostat or vibratome sections can be used. 4- Reduction step: Sections are reduced with the solution C containing sodium borohydride (0.1M) for 10 min. Then, the sections are washed 4 times with solution C without sodium borohydride. Application of anti-conjugated Dopamine antibodies: The final dilution is 1/1,000 to 1/5,000 in solution C containing triton X100 0.1%, plus 2% of non-specific serum. A dozen of sections can be incubated with 2ml of antibody solution overnight at 4°C. Then, after this period, the sections are washed 3 times (10 min) with solution C. N.B.: Antibodies may be used at a higher dilution. The customer should explore the antibody dilution to reduce the possibility of high background. Note that a substitution in the buffer system as used in our protocol may change the background and the antibody recognition. 5- PAP procedure: Second antibody: Sections are incubated with 1/100 dilution of goat anti-rabbit in solution C for 3 hours at 20°C or 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C PAP: Sections are incubated with 1/1,000 dilution of rabbit peroxidase anti-peroxidase complex in solution C for 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C Revelation: Antibody-antigen complexes are revealed using diaminobenzidine (25mg/100ml) (or other chromogen) dissolved in Tris 0.05M and filtrated 0.05% of H 2 O 2 is added. The sections are incubated for 10 min at 20°C. Reaction is stopped by transfering sections in 5ml of Tris 0.05M. Gemacbio sells the same antibodies raised in mouse (or rat): used together, these tools could be helpful for immunocytochemistry double labelling. Simultaneous detection of Tryptamine and Dopamine (DA) in rat brain 1- Perfusion: The rat will be deeply anesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the 500ml of 0.5M glutaraldehyde (G) solution in 0.1M cacodylate buffer pH 7,5 containing 0.9% sodium metabisulfite (SMB). 2- Post fixation: The brain will be removed , post-fixed for 60min in 0.5M glutaraldehyde (G) solution containing 0.9% sodium metabisulfite (SMB) and then washed thoroughly with 0.05M Tris buffer containing 0.05M SMB (Tris-SMB) pH 7,5. 3- Tissue sectionning: Vibratome sections will be cut through the region from the substantia to the raphe nuclei for the molecular detection of dopamine (DA) and tryptamine (T) . 4- Reduction step: Sections will be placed in Tris-SMB buffer, reduced using 0.1M sodium borohydride (in the same buffer), washed and then processed for immunocyto-chemistry using the peroxide / anti-peroxide (PAP) method. 5- Primary antibody: Following incubation with 3% non-specific serum in Tris-SMB for 1h at room temperature, the sections are incubated overnight at 4°C in Tris-SMB buffer containing 1% non specific serum, 0.02% Triton X100, diluted antibodies against DA and T (final dilutions 1/10,000). The T antiserum had previously been purified on the glutaraldehyde- conjugated protein carriers used during immunization and a monoclonal antibody to conjugated DA was produced. Recommended dilutions for Immunocytochemistry (1/1,000-1/5,000) Recommended dilutions for Western Blot (1/1,000-1/2,000)
Restrictions For Research Use only
Format Lyophilized
Storage 4 °C